EP2582829B1

EP2582829B1 - Methods for improved c4-dicarboxylic acid production in filamentous fungi

Info

Publication number: EP2582829B1
Application number: EP11728507.2A
Authority: EP
Inventors: Sheryl Luttringer; Debbie Yaver
Original assignee: Novozymes Inc
Current assignee: Novozymes Inc
Priority date: 2010-06-21
Filing date: 2011-06-21
Publication date: 2016-11-30
Anticipated expiration: 2031-06-21
Also published as: US20130288321A1; CN102947458A; WO2011163270A1; IN2013CN00459A; US20110312046A1; EP2582829A1; BR112012028031A2; CN102947458B; US8497103B2

Description

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background of the Invention

Field of the Invention

The present invention relates to methods for improving the production of C4-dicarboxylic acids (e.g., malic acid) in filamentous fungi.

Description of the Related Art

Organic acids have a long history of commercial use in a variety of industries. For example, organic acids are used in the food and feed industries (citric acid, ascorbic acid, lactic acid, acetic acid, and gluconic acid) as monomers for the production of various polymers (adipic acid, lactic acid, acrylic acid, and itaconic acid), as metal chelators (gluconic acid), and as "green" solvents (acetic acid) (Sauer et al., 2008, Trends in Biotechnology 26: 100-108). Organic acids may themselves be commercial products or they may be chemical building blocks used in the manufacture of other chemicals. In addition to specialty applications, it has long been recognized that C4-dicarboxylic acids can also serve as building block compounds for the production of large volume industrial chemicals, such as 1,4-butanediol, tetrahydrofuran, and gamma-butyrolactone. The cost of producing these large volume industrial chemicals by traditional petrochemical routes has increased significantly due to the high cost of petroleum derived building blocks.
Organic acids are produced commercially either by chemical synthesis from petroleum derived feedstocks (e.g., fumaric acid, malic acid, acrylic acid, and adipic acid) or by microbial fermentation (e.g., citric acid, lactic acid, gluconic acid, and itaconic acid). Some organic acids such as fumaric acid and malic acid can also be produced by microbial fermentation, but are currently produced commercially by chemical synthesis from petrochemical feedstocks due to lower production costs. However, the rising cost of petroleum derived building block chemicals, the geopolitical instability affecting crude oil prices, and the desire to implement manufacturing processes that utilize feedstocks derived from renewable resources have stimulated a renewed interest in producing organic acids and other chemicals by microbial fermentation.
While malic acid is produced commercially today by chemical synthesis from petrochemical feedstocks, it can also be produced by microbial fermentation. Malic acid has been produced at high levels in genetically engineered yeast (Saccharomyces cerevisiae) (Zelle et al., 2008, Appl. Environ. Microbiol. 74: 2766-2777) and naturally occurring filamentous fungi such as Aspergillus spp. ( U.S. Patent No. 3,063,910 ; Bercovitz et al., 1990, Appl. Environ. Microbiol. 56: 1594-1597). Abe et al. ( U.S. Patent No. 3,063,910 ) and Bercovitz et al. (1990, Appl. Environ. Microbiol. 56: 1594-1597) reported high levels of malic acid production in several species of Aspergillus. Moreover, Battat et al. (1991, Biotechnol. Bioengineering, 37: 1108-1116) reported malic acid production as high as 113 g/L by Aspergillus flavus in a stirred fermentor under optimized conditions. Dicarboxylic acid production by microbial fermentation in yeast is described in WO 2010/003728 . Malic acid production by microbial fermentation is also described in WO 2009/011974 and WO 2009/155382 . Improvement of malic acid production by genetic engineering of Aspergillus will enable economical commercial malic acid production by fermentation.
Malic acid overproduction in Aspergillus spp. occurs under specific culture conditions (aerobic conditions and high C:N ratio; calcium carbonate is also added as a neutralizing agent and as source of CO₂ for malic acid biosynthesis). Under these conditions, overflow metabolism via the cytosolic, reductive tricarboxylic acid (TCA) cycle results in increased malic acid biosynthesis and secretion into the culture medium. Increased malic acid production has been reported in Saccharomyces cerevisiae by increasing the level of pyruvate carboxylase (Bauer et al., 1999, FEMS Microbiol Lett. 179: 107-113) or malate dehydrogenase (Pines et al., 1997, Appl. Microbiol. Biotechnol. 48: 248-255) using genetic engineering and increasing expression of a malic acid transporter (Zelle et al., 2008, supra). It has been suggested, based on biochemical evidence, that malate dehydrogenase activity is limiting malic acid production in Aspergillus flavus strain ATCC 13697 (Peleg et al., 1988, Appl. Microbiol. Biotechnol. 28: 69-75). PCT Application No. PCT/US10/47002 , entitled "Methods for Improving Malic Acid Production in Filamentous Fungi" filed August 27, 2010, the content of which is hereby incorporated by reference in its entirety, describes malic acid production in filamentous fungi.
It would be advantageous in the art to improve C4-dicarboxylic acid production, such as malic acid production, in Aspergillus as a result of genetic engineering using recombinant DNA techniques. The present invention provides, inter alia, methods for improving C4-dicarboxylic acid production (e.g., malic acid production).

Summary of the Invention

The present invention also relates to an Aspergillus oryzae host cell comprising a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter, wherein the transporter is selected from:

(a) a C4-dicarboxylic acid transporter having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2 or 4;
(b) a C4-dicarboxylic acid transporter encoded by a polynucleotide that hybridizes under high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, 3, or the full-length complementary strand thereof; and
(c) a C4-dicarboxylic acid transporter encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or 3,

Aspergillus

The invention also relates to methods of producing or increasing C4-dicarboxylic acid production using an Aspergillus oryzae host cell of the invention.

Brief Description of the Figures

Figure 1 shows a restriction map of pAcC4T.
Figure 2 shows a restriction map of pShTh60.
Figure 3 shows a restriction map of pShTh120AcC4T.
Figure 4 shows the genomic DNA sequence and the deduced amino acid sequence of an Aspergillus clavatus C4-dicarboxylic acid transporter gene (SEQ ID NOs: 1 and 2, respectively).
Figure 5 shows a restriction map of pAfC4T.
Figure 6 shows a restriction map of pShTh121AfC4T.
Figure 7 shows the genomic DNA sequence and the deduced amino acid sequence of an Aspergillus fumigates C4-dicarboxylic acid transporter gene (SEQ ID NOs: 3 and 4, respectively).
Figure 8 shows the genomic DNA sequence and the deduced amino acid sequence of an Aspergillus oryzae NRRL 3488 malate dehydrogenase gene (mdh3) (SEQ ID NOs: 11 and 12, respectively).
Figures 9A and 9B together show the genomic DNA sequence and the deduced amino acid sequence of an Aspergillus oryzae NRRL 3488 pyruvate carboxylase gene (pyc) (SEQ ID NOs: 13 and 14, respectively).

Definitions

C4-dicarboxylic acid transporter: The term "C4-dicarboxylic acid transporter" is defined herein as a dicarboxylic acid permease that can transport malic acid, succinic acid, oxaloacetic acid, malonic acid, and/or fumaric acid outside a cell (Grobler et al., 1995, Yeast 11: 1485-1491; Camarasa et al., 2001, Applied and Environmental Microbiology 67: 4144-4151). A computational method to predict mitochondrially imported proteins and their targeting sequences is described by Claros and Vincens, 1996, Eur. J. Biochem. 241: 779-786.
In some aspects, the C4-dicarboxylic acid transporters have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the C4-dicarboxylic acid transporter activity (e.g., malic acid transporter activity) of the mature polypeptide sequence of SEQ ID NO: 2 or the mature polypeptide sequence of SEQ ID NO: 4.
Malate dehydrogenase: The term "malate dehydrogenase" is defined herein as a malate:NAD⁺ oxidoreductase (EC 1.1.1.37) that catalyzes the reduction of oxaloacetate in the presence of NADH + H⁺ to malate and NAD⁺. For purposes of the present invention, malate dehydrogenase activity is determined according to the following procedure. The assay solution consists of 1 mM oxaloacetic acid, 100 mM Tris pH 8.0, 10 mM NaHCO₃, 5 mM MgCl₂, and 0.1 mM NADH (Sigma Chemical Co., St. Louis, MO, USA). The assay solution without oxaloacetic acid as substrate is run as a control to measure background NADH degradation rates. Dilutions of 1/100, 1/500, 1/2500, and 1/12500 of each supernatant are prepared with double-distilled water. Aliquots of 270 µl of the assay solution are dispensed into 96 well polystyrene flat bottom plates. A 30 µl sample of each diluted supernatant is added to initiate the assay. The reactions are monitored using a SPECTRAMAX® 340PC plate reader (Molecular Devices, Sunnyvale, CA, USA) with the following settings: 340 nm, kinetic reading. A concentration series of NADH is used to construct a standard curve and a dilution series of purified malic dehydrogenase (Sigma Chemical Co., St. Louis, MO, USA) is used as a positive control. One unit of malate dehydrogenase activity equals the amount of enzyme capable of converting 1 µmole of oxaloacetate and NADH + H⁺ to malate and NAD⁺ per minute at pH 8.0, 25°C.
In some aspects, the malate dehydrogenases have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the malate dehydrogenase activity of the mature polypeptide sequence of SEQ ID NO: 12.
Pyruvate carboxylase: The term "pyruvate carboxylase" is defined herein as a pyruvate:carbon-dioxide ligase (ADP-forming) (EC 6.4.1.1) that catalyzes the carboxylation of pyruvate in the presence of ATP and HCO₃ ^- to oxaloacetate, ADP, and phosphate. For purposes of the present invention, pyruvate carboxylase activity is determined according to the procedure of the SIGMA® Quality Control Test procedure for pyruvate carboxylase (Sigma Chemical Co., St. Louis, MO, USA) except the assay uses Tris buffer at pH 8.0. One unit of pyruvate carboxylase activity equals the amount of enzyme capable of converting 1 µmole of pyruvate and CO₂ to oxaloacetate per minute at pH 7.8, 30°C.
In some aspects, the pyruvate carboxylases have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the pyruvate carboxylase activity of the mature polypeptide sequence of SEQ ID NO: 14.
Heterologous polynucleotide: The term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell; a native polynucleotide in which structural modifications have been made to the coding region; a native polynucleotide whose expression is quantitatively altered as a result of a manipulation of the DNA by recombinant DNA techniques, e.g., a different (foreign) promoter; or a native polynucleotide whose expression is quantitatively altered by the introduction of one or more (e.g., two, several) extra copies of the polynucleotide into the host cell.
Isolated/purified: The terms "isolated" and "purified" mean a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated. For example, a polypeptide may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, at least 93% pure, at least 95% pure, at least 97%, at least 98% pure, or at least 99% pure, as determined by SDS-PAGE and a polynucleotide may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90%, at least 93% pure, at least 95% pure, at least 97%, at least 98% pure, or at least 99% pure, as determined by agarose electrophoresis.
Coding sequence: The term "coding sequence" means a polynucleotide sequence, which specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a sequence of genomic DNA, cDNA, a synthetic polynucleotide, and/or a recombinant polynucleotide.
cDNA sequence: The term "cDNA sequence" means a sequence of DNA following reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. The initial, primary RNA transcript from genomic DNA is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA. A cDNA sequence lacks intervening intron sequences that may be present in the corresponding genomic DNA sequence. Accordingly, the phrase "the cDNA sequence of SEQ ID NO: X" intends the resulting sequence after the intervening intron sequences of SEQ ID NO: X, if present, are removed. In some instances-when a referenced genomic DNA sequence lacks intervening intron sequences-a cDNA sequence may be identical to its corresponding genomic DNA sequence.
Genomic DNA sequence: The term "genomic DNA sequence" means a DNA sequence found in the genome of a source organism (e.g., a eukaryotic or prokaryotic genome). In some instances, a genomic DNA sequence from a eukaryotic genome contains one or more intervening intron sequences that are removed from the primary RNA transcript as a result of RNA splicing. Accordingly, the phrase "the genomic DNA sequence of SEQ ID NO: Y" intends the corresponding DNA sequence from the source organism which includes intervening intron sequences, if any, that are present before RNA splicing.
Mature polypeptide sequence: The term "mature polypeptide sequence" means the portion of the referenced polypeptide sequence after any post-translational sequence modifications (such as N-terminal processing and/or C-terminal truncation). In some instances, the mature polypeptide sequence may be identical to the entire referenced polypeptide sequence. In one aspect, the mature polypeptide sequence is amino acids 53 to 392 of SEQ ID NO: 2 based on the Vector NIT® program (Invitrogen, CA, USA) that predicts amino acids 1 to 52 of SEQ ID NO: 2 are a signal peptide. In another aspect, the mature polypeptide sequence is amino acids 1 to 393 of SEQ ID NO: 4.
Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means the portion of the referenced polynucleotide sequence (e.g., genomic or cDNA sequence) that encodes a mature polypeptide sequence. In some instances, the mature polypeptide coding sequence may be identical to the entire referenced polynucleotide sequence. In one aspect, the mature polypeptide coding sequence is nucleotides 157 to 1179 of SEQ ID NO: 1 based on the Vector NIT® program (Invitrogen, CA, USA) that predicts nucleotides 1 to 156 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 1 to 1182 of SEQ ID NO: 3.
Fragment: The term "fragment" means a polypeptide having one or more (e.g., two, several) amino acids deleted from the amino and/or carboxyl terminus of a referenced polypeptide sequence. In one aspect, the fragment has C4-dicarboxylic acid transporter activity. In another aspect, a fragment contains at least 332 amino acid residues, e.g., at least 352 amino acid residues or at least 372 amino acid residues of SEQ ID NO: 2. In another aspect, a fragment contains at least 332 amino acid residues, e.g., at least 352 amino acid residues or at least 372 amino acid residues of SEQ ID NO: 4.
Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., two, several) nucleotides deleted from the 5' and/or 3' end of the referenced nucleotide sequence. In one aspect, the subsequence encodes a fragment having C4-dicarboxylic acid transporter activity. In another aspect, a subsequence contains at least 996 nucleotides, e.g., at least 1056 nucleotides or at least 1116 nucleotides of SEQ ID NO: 1. In another aspect, a subsequence contains at least 996 nucleotides, e.g., at least 1056 nucleotides or at least 1116 nucleotides of SEQ ID NO: 3.
Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule-single-stranded or double-stranded-which is isolated from a naturally occurring gene, modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, or synthetic, wherein the nucleic acid molecule comprises one or more (e.g., two, several) control sequences.
Control sequence: The term "control sequence" means a nucleic acid sequence necessary for polypeptide expression. Control sequences may be native or foreign to the polynucleotide encoding the polypeptide, and native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, and transcription terminator sequence. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. At a minimum, the expression vector comprises a promoter sequence, and transcriptional and translational stop signal sequences.
Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention (e.g., a polynucleotide encoding a C4-dicarboxylic acid transporter). The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Variant: The term "variant" means a polypeptide having activity, e.g., C4-dicarboxylic acid transporter activity, comprising an alteration, i.e., a substitution, insertion, and/or deletion of one or more (e.g., two, several) amino acid residues at one or more positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding one or more, e.g., 1-3 amino acids, adjacent to an amino acid occupying a position.
Volumetric productivity: The term "volumetric productivity" refers to the amount of referenced product produced (e.g., the amount of a C4-dicarboxylic acid produced) per volume of the system used (e.g., the total volume of media and contents therein) per unit of time.
Fermentable medium: The term "fermentable medium" refers to a medium comprising one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides, wherein the medium is capable, in part, of being converted (fermented) by a host cell into a desired product, such as a C4-dicarboxylic acid. In some instances, the fermentation medium is derived from a natural source, such as sugar cane, starch, or cellulose, and may be the result of pretreating the source by enzymatic hydrolysis (saccharification).
Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X".
As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that the aspects of the invention described herein include "consisting" and/or "consisting essentially of" aspects.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Detailed Description of the Invention

The present disclosure describes the overexpression of specific genes in a host cell, such as a filamentous fungus (e.g., Aspergillus) to enhance the production of C4-dicarboxylic acids, (e.g., malic acid) that encompasses transport of the C4-dicarboxylic acid out of the cell via a C4-dicarboxylic acid transporter. In one aspect, the C4-dicarboxylic acid transporter is a transporter that is overexpressed under culture conditions that produces C4-dicarboxylic acid in high titers. The recombinant host cell may further comprise a heterologous polynucleotide encoding a malate dehydrogenase and/or a heterologous polynucleotide encoding a pyruvate carboxylase.

C4-Dicarboxylic Acid Transporters and Polynucleotides Encoding C4-Dicarboxylic Acid Transporters

In one aspect of the recombinant host cells and methods described herein, the C4-dicarboxylic acid transporter is selected from: (a) a C4-dicarboxylic acid transporter having at least 90% sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof; (b) a C4-dicarboxylic acid transporter encoded by a polynucleotide that hybridizes under high stringency conditions with SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing; (c) a C4-dicarboxylic acid transporter encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing; (d) a C4-dicarboxylic acid transporter variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has C4-dicarboxylic acid transporter activity.
In one aspect, the C4-dicarboxylic acid transporter comprises or consists of an amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof. In one aspect, the C4-dicarboxylic acid transporter comprises an amino acid sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof.
In one aspect, the C4-dicarboxylic acid transporter comprises or consists of an amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2 or the mature polypeptide sequence thereof. In one aspect, the C4-dicarboxylic acid transporter comprises an amino acid sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from SEQ ID NO: 2 or the mature polypeptide sequence thereof. In another aspect, the C4-dicarboxylic acid transporter comprises an amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or the mature polypeptide sequence thereof. In one aspect, the C4-dicarboxylic acid transporter comprises an amino acid sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from SEQ ID NO: 4 or the mature polypeptide sequence thereof.
In one aspect, the C4-dicarboxylic acid transporter comprises or consists of the amino acid sequence of SEQ ID NO: 2, the mature polypeptide sequence of SEQ ID NO: 2, an allelic variant thereof, or a fragment of the foregoing, having C4-dicarboxylic acid transporter activity. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of the mature polypeptide sequence of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of amino acids 1 to 392 of SEQ ID NO: 2.
In one aspect, the C4-dicarboxylic acid transporter comprises or consists of the amino acid sequence of SEQ ID NO: 4, the mature polypeptide sequence of SEQ ID NO: 4, an allelic variant thereof, or a fragment of the foregoing, having C4-dicarboxylic acid transporter activity. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of the mature polypeptide sequence of SEQ ID NO: 4. In another aspect, the C4-dicarboxylic acid transporter comprises or consists of amino acids 1 to 393 of SEQ ID NO: 4.
In one aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide that hybridizes under high stringency conditions, or very high stringency conditions with SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
In one aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide that hybridizes under high stringency conditions, or very high stringency conditions with SEQ ID NO: 1, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing. In another aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide that hybridizes under at high stringency conditions, or very high stringency conditions with SEQ ID NO: 3, the mature polypeptide coding sequence thereof, of the full-length complementary strand of the foregoing.
In one aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing.
In one aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing.
In one aspect, the C4-dicarboxylic acid transporter is encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing.
In one aspect, the C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 1 or 3, or the mature polypeptide coding sequence thereof. In one aspect, the C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 1 or the mature polypeptide coding sequence thereof. In one aspect, the C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 1. In one aspect, the C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 3 or the mature polypeptide coding sequence thereof. In one aspect, the C4-dicarboxylic acid transporter is encoded by SEQ ID NO: 3. In one aspect, the C4-dicarboxylic acid transporter is encoded by a subsequence of SEQ ID NO: 1 or 3, wherein the subsequence encodes a polypeptide having C4-dicarboxylic acid transporter activity. In one aspect, the C4-dicarboxylic acid transporter is encoded by a subsequence of SEQ ID NO: 1, wherein the subsequence encodes a polypeptide having C4-dicarboxylic acid transporter activity. In one aspect, the C4-dicarboxylic acid transporter is encoded by a subsequence of SEQ ID NO: 3, wherein the subsequence encodes a polypeptide having C4-dicarboxylic acid transporter activity.
In one aspect, the C4-dicarboxylic acid transporter is a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof. In one aspect, the C4-dicarboxylic acid transporter is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO: 2. In one aspect, the C4-dicarboxylic acid transporter is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of the mature polypeptide sequence of SEQ ID NO: 2. In one aspect, the C4-dicarboxylic acid transporter is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO: 4. In one aspect, the C4-dicarboxylic acid transporter is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of the mature polypeptide sequence of SEQ ID NO: 4.
Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/IIe, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/IIe, Leu/Val, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for activity (e.g., C4-dicarboxylic acid transporter activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the referenced parent polypeptide.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413 ; or WO 95/22625 . Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409 ; WO 92/06204 ), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
In some aspects, the total number of amino acid substitutions, deletions and/or insertions of SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof, is not more than 10, e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8 or 9.
In another aspect, the C4-dicarboxylic acid transporter is a fragment of SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof, wherein the fragment has C4-dicarboxylic acid transporter activity. In one aspect, the C4-dicarboxylic acid transporter is a fragment of SEQ ID NO: 2 or the mature polypeptide sequence thereof, wherein the fragment has C4-dicarboxylic acid transporter activity. In one aspect, the fragment contains at least 332 amino acid residues, e.g., at least 352 amino acid residues, or at least 372 amino acid residues of SEQ ID NO: 2. In one aspect, the fragment contains a C4-dicarboxylic acid transporter domain, e.g., the putative transporter domain of amino acids 39 to 337 of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic acid transporter is a fragment of SEQ ID NO: 4 or the mature polypeptide sequence thereof, wherein the fragment has C4-dicarboxylic acid transporter activity. In one aspect, the fragment contains at least 332 amino acid residues, e.g., at least 352 amino acid residues, or at least 372 amino acid residues of SEQ ID NO: 4. In one aspect, the fragment contains a C4-dicarboxylic acid transporter domain, e.g., the putative transporter domain of amino acids 41 to 338 of SEQ ID NO: 4.
The C4-dicarboxylic acid transporter may be a fused polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fused polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).
A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Techniques used to isolate or clone a polynucleotide-such as a polynucleotide encoding a C4-dicarboxylic acid transporter-as well as any other polypeptide used in any of the aspects mentioned herein, are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shares structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Aspergillus, or another or related organism, and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.
The polynucleotide of SEQ ID NO: 1 or 3, or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2 or 4; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding a C4-dicarboxylic acid transporter from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, e.g., at least 14 nucleotides, at least 25 nucleotides, at least 35 nucleotides, at least 70 nucleotides in lengths. The probes may be longer, e.g., at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides in lengths. Even longer probes may be used, e.g., at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having C4-dicarboxylic acid transporter activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1 or 3, or a subsequence thereof, the carrier material is preferably used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to SEQ ID NO: 1 or 3, the mature polypeptide coding sequence of SEQ ID NO: 1 or 3, or the full-length complementary strand thereof, or a subsequence of the foregoing; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.
In one aspect, the nucleic acid probe is SEQ ID NO: 1 or 3. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1 or 3. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is SEQ ID NO: 1. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another aspect, the nucleic acid probe is SEQ ID NO: 3. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or a fragment thereof. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 4 or a fragment thereof.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C (very low stringency), at 50°C (low stringency), at 55°C (medium stringency), at 60°C (medium-high stringency), at 65°C (high stringency), and at 70°C (very high stringency).
For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization and hybridization at about 5°C to about 10°C below the calculated T_m using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mL following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed once in 6X SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T_m.
The C4-dicarboxylic acid transporter of the present invention may be obtained from a microorganism of any genus. As used herein, the term "obtained from" in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a cell in which the polynucleotide from the source has been inserted.
The C4-dicarboxylic acid transporter may be a bacterial C4-dicarboxylic acid transporter. For example, the C4-dicarboxylic acid transporter may be a Gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus C4-dicarboxylic acid transporter, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma C4-dicarboxylic acid transporter.
In one aspect, the C4-dicarboxylic acid transporter is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis C4-dicarboxylic acid transporter.
In another aspect, the C4-dicarboxylic acid transporter is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus C4-dicarboxylic acid transporter.
In another aspect, the C4-dicarboxylic acid transporter is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans C4-dicarboxylic acid transporter.
The C4-dicarboxylic acid transporter may be a fungal C4-dicarboxylic acid transporter. In one aspect, the fungal C4-dicarboxylic acid transporter is a yeast C4-dicarboxylic acid transporter such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia C4-dicarboxylic acid transporter.
In another aspect, the fungal C4-dicarboxylic acid transporter is a filamentous fungal C4-dicarboxylic acid transporter such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria C4-dicarboxylic acid transporter.
In another aspect, the C4-dicarboxylic acid transporter is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis C4-dicarboxylic acid transporter.
In another aspect, the C4-dicarboxylic acid transporter is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus clavatus, Aspergillus awamori, Aspergillus flavus, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride C4-dicarboxylic acid transporter.
In one aspect, the C4-dicarboxylic acid transporter is an Aspergillus C4-dicarboxylic acid transporter, such as an Aspergillus clavatus C4-dicarboxylic acid transporter or an Aspergillus fumigatus C4-dicarboxylic acid transporter. In one aspect, the C4-dicarboxylic acid transporter an Aspergillus clavatus C4-dicarboxylic acid transporter of SEQ ID NO: 2. In another aspect, the C4-dicarboxylic acid transporter an Aspergillus fumigatus C4-dicarboxylic acid transporter of SEQ ID NO: 4.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The C4-dicarboxylic acid transporter may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding a C4-dicarboxylic acid transporter may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a C4-dicarboxylic acid transporter has been detected with suitable probe(s) as described herein, the sequence may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

Malate Dehydrogenases and Polynucleotides Encoding Malate Dehydrogenases

In some aspects of the recombinant host cells and methods of use thereof, the host cells have malate dehydrogenase activity. In some aspects, the host cells comprise a heterologous polynucleotide encoding a malate dehydrogenase. The malate dehydrogenase can be any malate dehydrogenase that is suitable for practicing the invention. In one aspect, the malate dehydrogenase is an enzyme that is present in the cytosol of the host cell.
In one aspect of the recombinant host cells and methods described herein, the malate dehydrogenase is (a) a malate dehydrogenase having at least 60% sequence identity to SEQ ID NO: 12 or the mature polypeptide sequence thereof; (b) a malate dehydrogenase encoded by a polynucleotide that hybridizes under low stringency conditions with (i) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii); (c) a malate dehydrogenase encoded by a polynucleotide having at least 60% sequence identity to (iv) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v); (d) a malate dehydrogenase variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 12 or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has malate dehydrogenase activity.
In one aspect, the malate dehydrogenase comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 or the mature polypeptide sequence thereof. In one aspect, the malate dehydrogenase comprises an amino acid sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from SEQ ID NO: 12 or the mature polypeptide sequence thereof.
In one aspect, the malate dehydrogenase comprises or consists of the amino acid sequence of SEQ ID NO: 12, the mature polypeptide sequence of SEQ ID NO: 12, an allelic variant thereof, or a fragment of the foregoing, having malate dehydrogenase activity. In another aspect, the malate dehydrogenase comprises or consists of the amino acid sequence of SEQ ID NO: 12. In another aspect, the malate dehydrogenase comprises or consists of the mature polypeptide sequence of SEQ ID NO: 12. In another aspect, the malate dehydrogenase comprises or consists of amino acids 1 to 330 of SEQ ID NO: 12.
In one aspect, the malate dehydrogenase is encoded by a polynucleotide that hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, supra).
In one aspect, the malate dehydrogenase is encoded by a polynucleotide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (iv) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v).
In one aspect, the malate dehydrogenase is encoded by SEQ ID NO: 11, or the mature polypeptide coding sequence thereof. In one aspect, the malate dehydrogenase is encoded by SEQ ID NO: 11. In one aspect, the malate dehydrogenase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 11. In one aspect, the malate dehydrogenase is encoded by a subsequence of SEQ ID NO: 11, wherein the subsequence encodes a polypeptide having malate dehydrogenase activity. In one aspect, the subsequence contains at least 885 nucleotides, e.g., at least 930 nucleotides or at least 975 nucleotides of SEQ ID NO: 11.
In one aspect, the malate dehydrogenase is a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 12, or the mature polypeptide sequence thereof, as described supra. In one aspect, the malate dehydrogenase is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO: 12. In one aspect, the malate dehydrogenase is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of the mature polypeptide sequence of SEQ ID NO: 12. In some aspects, the total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide sequence of SEQ ID NO: 12 or the mature polypeptide sequence thereof is not more than 10, e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8 or 9.
In another aspect, the malate dehydrogenase is a fragment of SEQ ID NO: 12, or the mature polypeptide sequence thereof, wherein the fragment has malate dehydrogenase activity. In one aspect, the fragment contains at least 295 amino acid residues, e.g., at least 310 amino acid residues, or at least 325 amino acid residues of SEQ ID NO: 12.
The malate dehydrogenase may also be an allelic variant or artificial variant of a malate dehydrogenase.
The malate dehydrogenase can also include fused polypeptides or cleavable fusion polypeptides, as described supra.
Techniques used to isolate or clone a polynucleotide encoding a malate dehydrogenase are described supra.
The polynucleotide of SEQ ID NO: 11; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 12; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding malate dehydrogenases from strains of different genera or species, as described supra. Such probes are encompassed by the present invention. A genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridizes with the probes described above and encodes a malate dehydrogenase, as described supra.
In one aspect, the nucleic acid probe is SEQ ID NO: 11. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 11. In another aspect, the nucleic acid probe is a polynucleotide sequence that encodes SEQ ID NO: 12, the mature polypeptide sequence thereof, or a fragment of the foregoing.
For long probes of at least 100 nucleotides in length, very low to very high stringency and washing conditions are defined as described supra. For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency and washing conditions are defined as described supra.
The malate dehydrogenase may be obtained from microorganisms of any genus. In one aspect, the malate dehydrogenase may be a bacterial, a yeast, or a filamentous fungal malate dehydrogenase obtained from the microorganisms described herein. In another aspect, the malate dehydrogenase is an Aspergillus oryzae malate dehydrogenase, e.g., the Aspergillus oryzae malate dehydrogenase of SEQ ID NO: 12.
Other malate dehydrogenases that can be used to practice the present invention include, but are not limited to, a Aspergillus nidulans malate dehydrogenase (AN6717.1; SIMS et al., 2004, Mycol. Res. 108 : 853-857); Aspergillus niger malate dehydrogenase (An16g00120; Pel et al., 2007, Nature Biotechnology 25: 221-231); Phytophthora infestans malate dehydrogenase (PITG 13614.1; Calcagno et al., 2009, Mycological Research 113: 771-781); Saccharomyces cerevisiae malate dehydrogenase (YKL085W; McAlister-Henn and Thompson, 1987, J Bacteriol. 169: 5157-5166); Talaromyces emersonii malate dehydrogenase (AF439996, AF487682; Maloney et al., 2004, Eur. J. Biochem. 271: 3115-3126); and Ustilago maydis malate dehydrogenase (um00403, um11161; McCann and Snetselaar, 2008, Fungal Genetics and Biology 45: S77-S87), the Aspergillus oryzae malate dehydrogenase of SEQ ID NO: 16 (encoded by the polynucleotide sequence of SEQ ID NO: 15; see U.S. Application No. 12/870,523 , entitled "Methods for Improving Malic Acid Production in Filamentous Fungi" filed August 27, 2010), or any aspect of the malate dehydrogenase described in the respective reference therein.
The invention embraces any aspect of sequence identity, hybridization, variants and fragments described herein as applied to other malate dehydrogenase polypeptide sequences and polynucleotide sequences described above. For example, in one aspect, the malate dehydrogenase is (a) a malate dehydrogenase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16, or the mature polypeptide sequence thereof; (b) a malate dehydrogenase encoded by a polynucleotide that hybridizes under low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) SEQ ID NO: 15 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 15 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of the (i) or (ii); (c) a malate dehydrogenase encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (iv) SEQ ID NO: 15 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 15 or the mature polypeptide coding sequence thereof, or (vi) the full-length complementary strand of the (iv) or (v); (d) a malate dehydrogenase variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 16 or the mature polypeptide sequence thereof; or (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has malate dehydrogenase activity.
The malate dehydrogenase may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) as described supra.

Pyruvate Carboxylases and Polynucleotides Encoding Pyruvate Carboxylases

In some aspects of the recombinant host cells and methods of use thereof, the host cells have pyruvate carboxylase activity. In some aspects, the host cells comprise a heterologous polynucleotide encoding a pyruvate carboxylase. The pyruvate carboxylase can be any pyruvate carboxylase that is suitable for practicing the invention. In one aspect, the pyruvate carboxylase is an enzyme that is present in the cytosol of the host cell.
In one aspect of the recombinant host cells and methods described herein, the pyruvate carboxylase is (a) a pyruvate carboxylase having at least 60% sequence identity to SEQ ID NO: 14 or the mature polypeptide sequence thereof; (b) a pyruvate carboxylase encoded by a polynucleotide that hybridizes under low stringency conditions with (i) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii); (c) a pyruvate carboxylase encoded by a polynucleotide having at least 60% sequence identity to (iv) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v); (d) a pyruvate carboxylase variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 14 or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has pyruvate carboxylase activity.
In one aspect, the pyruvate carboxylase comprises or consists of an amino acid sequence having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, or the mature polypeptide sequence thereof. In one aspect, the pyruvate carboxylase comprises an amino acid sequence that differs by no more than ten amino acids, e.g., by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by one amino acid from SEQ ID NO: 14 or the mature polypeptide sequence thereof.
In one aspect, the pyruvate carboxylase comprises or consists of the amino acid sequence of SEQ ID NO: 14, the mature polypeptide sequence of SEQ ID NO: 14, an allelic variant thereof, or a fragment of the foregoing, having pyruvate carboxylase activity. In another aspect, the pyruvate carboxylase comprises or consists of the amino acid sequence of SEQ ID NO: 14. In another aspect, the pyruvate carboxylase comprises or consists of the mature polypeptide sequence of SEQ ID NO: 14. In another aspect, the pyruvate carboxylase comprises or consists of amino acids 1 to 1193 of SEQ ID NO: 14.
In one aspect, the pyruvate carboxylase is encoded by a polynucleotide that hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, supra).
In one aspect, the pyruvate carboxylase is encoded by a polynucleotide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to (iv) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v).
In one aspect, the pyruvate carboxylase is encoded by SEQ ID NO: 13 or the mature polypeptide coding sequence thereof. In one aspect, the pyruvate carboxylase is encoded by SEQ ID NO: 13. In one aspect, the pyruvate carboxylase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 13. In one aspect, the pyruvate carboxylase is encoded by a subsequence of SEQ ID NO: 13, wherein the subsequence encodes a polypeptide having pyruvate carboxylase activity. In one aspect, the subsequence contains at least 3060 nucleotides, e.g., at least 3240 nucleotides or at least 3420 nucleotides of SEQ ID NO: 13.
In one aspect, the pyruvate carboxylase is a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 14, or the mature polypeptide sequence thereof, as described supra. In one aspect, the pyruvate carboxylase is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO: 14. In one aspect, the pyruvate carboxylase is a variant comprising a substitution, deletion, and/or insertion of one or more amino acids of the mature polypeptide sequence of SEQ ID NO: 14. In some aspects, the total number of amino acid substitutions, deletions and/or insertions of SEQ ID NO: 14 or the mature polypeptide sequence thereof is not more than 14, e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8 or 9.
In another aspect, the pyruvate carboxylase is a fragment of SEQ ID NO: 14, or the mature polypeptide sequence thereof, wherein the fragment has pyruvate carboxylase activity. In one aspect, the fragment contains at least 1020 amino acid residues, e.g., at least 1080 amino acid residues, or at least 1140 amino acid residues of SEQ ID NO: 14.
The pyruvate carboxylase may also be an allelic variant or artificial variant of a pyruvate carboxylase.
The pyruvate carboxylase can also include fused polypeptides or cleavable fusion polypeptides, as described supra.
The pyruvate carboxylase can also be a variant of a mitochondrial pyruvate carboxylase, such that in vivo importation into the mitochondria is reduced thereby increasing the level of the pyruvate carboxylase variant in the cytosol.
Techniques used to isolate or clone a polynucleotide encoding a pyruvate carboxylase are described supra.
The polynucleotide of SEQ ID NO: 13 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 14 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding pyruvate carboxylases from strains of different genera or species, as described supra. Such probes are encompassed by the present invention. A genomic DNA or cDNA library prepared from such other organisms may be screened for DNA that hybridizes with the probes described above and encodes a pyruvate carboxylase, as described supra.
In one aspect, the nucleic acid probe is SEQ ID NO: 13. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 13. In another aspect, the nucleic acid probe is a polynucleotide sequence that encodes SEQ ID NO: 14, the mature polypeptide sequence thereof, or a fragment of the foregoing.
For long probes of at least 100 nucleotides in length, very low to very high stringency and washing conditions are defined as described supra. For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency and washing conditions are defined as described supra.
The pyruvate carboxylase may be obtained from microorganisms of any genus. In one aspect, the pyruvate carboxylase may be a bacterial, a yeast, or a filamentous fungal pyruvate carboxylase obtained from the microorganisms described herein. In another aspect, the pyruvate carboxylase is an Aspergillus oryzae pyruvate carboxylase, e.g., the Aspergillus oryzae pyruvate carboxylase of SEQ ID NO: 14.
Other pyruvate carboxylases that can be used to practice the present invention include, but are not limited to, a Aspergillus clavatus NRRL 1 pyruvate carboxylase (XP_001271664; Direct Submission, Submitted (26-OCT-2006), The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA); Aspergillus fumigatus Af293 pyruvate carboxylase (XP_752054; Nierman et al., 2005, Nature 438: 1151-1156); Aspergillus nidulans FGSC A4 pyruvate carboxylase (XP_662066; Galagan et al., 2005, Nature 438: 1105-1115); Aspergillus niger pyruvate carboxylase (An15g02820; Pel et al., 2007, Nature Biotechnology 25: 221 - 231; ASPNG 5061; Panneman et al., Submitted (JUL-1998) to the EMBL/GenBank/DDBJ databases); Aspergillus terreus pyruvate carboxylase (093918; Direct Submission, Submitted (OCT-1998) The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA); Magnaporthe grisea 70-15 pyruvate carboxylase (XP_367852; Direct Submission, Submitted (26-SEP-2005) Broad Institute of MIT and Harvard, 320 Charles Street, Cambridge, MA 02142, USA); Neurospora crassa OR74A pyruvate carboxylase (XP_965636; Galagan et al., 2003, Nature 422: 859-868); Rhizopus oryzaepyruvate carboxylase (R03G_06931.1); Saccharomyces cerevisiae pyruvate carboxylase (NP_009777; Gaffeau et al., 1996, Science 274: 546-547); Schizosaccharomyces pombe pyruvate carboxylase (NP_595900; Direct Submission, Submitted (29-JUN-2007) European Schizosaccharomyces genome sequencing project, Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA); and Ustilago maydis pyruvate carboxylase (um01054; McCann and Snetselaar, 2008, Fungal Genetics and Biology 45: S77-S87). The invention embraces any aspect of sequence identity, hybridization, variants and fragments described herein as applied to the malate dehydrogenase polypeptide sequences and polynucleotide sequences described above.
The pyruvate carboxylase may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc,) as described supra.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a C4-dicarboxylic acid transporter (or other polynucleotides described herein, such as a polynucleotide encoding a malate dehydrogenase and/or a pyruvate carboxylase) linked to one or more (e.g., two, several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Such nucleic acid constructs may be used in any of the host cells and methods describe herein. In one aspect, the heterologous polynucleotide encoding a C4-dicarboxylic acid transporter is operably linked to promoter foreign to the polynucleotide. In one aspect, a second heterologous polynucleotide encoding a malate dehydrogenase is operably linked to promoter foreign to the polynucleotide. In one aspect, a third heterologous polynucleotide encoding a pyruvate carboxylase is operably linked to promoter foreign to the polynucleotide.
A polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter sequence, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a C4-dicarboxylic acid transporter or other polynucleotides described herein, such as a polynucleotide encoding a malate dehydrogenase and/or a pyruvate carboxylase). The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease ( WO 96/00787 ), Fusarium venenatum amyloglucosidase ( WO 00/56900 ), Fusarium venenatum Daria ( WO 00/56900 ), Fusarium venenatum Quinn ( WO 00/56900 ), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter from a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include modified promoters from the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof.
The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
The control sequence may also be a suitable leader sequence, when transcribed is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus nigeralpha-glucosidase.
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase ( WO 95/33836 ), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present at the N-terminus of a polypeptide, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant host cells and methods utilizing recombinant expression vectors comprising a polynucleotide encoding a C4-dicarboxylic acid transporter (or other polynucleotides described herein, such as a polynucleotide encoding a malate dehydrogenase and/or a pyruvate carboxylase), a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., two, several) convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
In one aspect, each polynucleotide encoding a C4-dicarboxylic acid transporter, a malate dehydrogenase, and/or a pyruvate carboxylase described herein is contained on an independent vector. In one aspect, two of the polynucleotides are contained on a single vector. In one aspect, all the polynucleotides encoding the C4-dicarboxylic acid transporter, the malate dehydrogenase, and the pyruvate carboxylase are contained on a single vector.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more (e.g., two, several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883 ). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883 .
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

As described herein, the present invention relates to, inter alia, recombinant Aspergillus oryzae host cells comprising a polynucleotide described herein (e.g., a polynucleotide encoding a C4-dicarboxylic acid transporter, a malate dehydrogenase, and/or a pyruvate carboxylase) operably linked to one or more (e.g., two, several) control sequences that direct the production of a polypeptides described herein for the recombinant production of a C4-dicarboxylic acid. The invention also embraces methods of using such host cells for the production of a C4-dicarboxylic acid. The host cell may comprise any one or combination of a plurality of the polynucleotides described. For example, in one aspect, the recombinant host cell comprises a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter; and optionally comprises a heterologous polynucleotide encoding a heterologous polynucleotide encoding a malate dehydrogenase, and/or a heterologous polynucleotide encoding pyruvate decarboxylase; wherein the host cell produces (or is capable of producing) a greater amount of a C4-dicarboxylic acid compared to the host cell without the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter when cultivated under the same conditions.
In one aspect, the recombinant host cell comprises:

(1) a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter, such as a C4-dicarboxylic acid transporter selected from: (a) a C4-dicarboxylic acid transporter having at least 90% sequence identity to SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof; (b) a C4-dicarboxylic acid transporter encoded by a polynucleotide that hybridizes under high stringency conditions with SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing; (c) a C4-dicarboxylic acid transporter encoded by a polynucleotide having at least 60% sequence identity to SEQ ID NO: 1 or 3, the mature polypeptide coding sequence thereof, or the full-length complementary strand of the foregoing; (d) a C4-dicarboxylic acid transporter variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 2 or 4, or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has C4-dicarboxylic acid transporter activity;
(2) an optional heterologous second polynucleotide encoding a malate dehydrogenase, such as a malate dehydrogenase selected from: (a) a malate dehydrogenase having at least 60% sequence identity to SEQ ID NO: 12 or the mature polypeptide sequence thereof; (b) a malate dehydrogenase encoded by a polynucleotide that hybridizes under low stringency conditions with (i) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii); (c) a malate dehydrogenase encoded by a polynucleotide having at least 60% sequence identity to (iv) SEQ ID NO: 11 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 11 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v); (d) a malate dehydrogenase variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 12 or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has malate dehydrogenase activity; and
(3) an optional heterologous third polynucleotide encoding a pyruvate carboxylase, such as a pyruvate carboxylase selected from: (a) a pyruvate carboxylase having at least 60% sequence identity to SEQ ID NO: 14 or the mature polypeptide sequence thereof; (b) a pyruvate carboxylase encoded by a polynucleotide that hybridizes under low stringency conditions with (i) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (ii) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, or (iii) the full-length complementary strand of (i) or (ii); (c) a pyruvate carboxylase encoded by a polynucleotide having at least 60% sequence identity to (iv) SEQ ID NO: 13 or the mature polypeptide coding sequence thereof, (v) the cDNA sequence of SEQ ID NO: 13 or the mature polypeptide coding sequence thereof; or (vi) the full-length complementary strand of (iv) or (v); (d) a pyruvate carboxylase variant comprising a substitution, deletion, and/or insertion of one or more (e.g., two, several) amino acids of SEQ ID NO: 14 or the mature polypeptide sequence thereof; and (e) a fragment of a polypeptide of (a), (b), (c), or (d) that has pyruvate carboxylase activity;

In one aspect, the host cell comprises a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter described herein (e.g., SEQ ID NO: 1 or 3, or any described aspect thereof) and a heterologous polynucleotide encoding a malate dehydrogenase. In the present invention, the malate dehydrogenase can be any malate dehydrogenase that is suitable for practicing the present invention, as described supra. In another aspect, the host cell comprises a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter described herein (e.g., SEQ ID NO: 1 or 3, or any described aspect thereof) and a heterologous polynucleotide encoding a pyruvate carboxylase. In the present invention, the pyruvate carboxylase can be any pyruvate carboxylase that is suitable for practicing the present invention, as described supra. In particular, the pyruvate carboxylase is preferably an enzyme that is present in the cytosol of the host cell. In one aspect, the host cell comprises a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter described herein (e.g., SEQ ID NO: 1 or 3, or any described aspect thereof), a second heterologous polynucleotide encoding a malate dehydrogenase, and a third heterologous polynucleotide encoding a pyruvate carboxylase.
A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The aspects described below apply to the host cells, per se, as well as methods using the host cells.
The fungal host cell is an Aspergillus oryzaecell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787 . Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
In some aspects, the host cell comprises one or more (e.g., two, several) polynucleotide(s) described herein, wherein the host cell secretes (and/or is capable of secreting) an increased level of C4-dicarboxylic acid compared to the host cell without the one or more polynucleotide(s) when cultivated under the same conditions. In some aspects, the host cell secretes and/or is capable of secreting an increased level of C4-dicarboxylic acid (e.g., malic acid) of at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at 500% compared to the host cell without the one or more polynucleotide(s) (e.g., without the heterologous polynucleotide encoding a C4-dicarboxylic acid transporter), when cultivated under the same conditions.
In any of the aspects of the recombinant host cells and methods described herein, the C4-dicarboxylic acid may be malic acid, succinic acid, oxaloacetic acid, malonic acid, or fumaric acid, or combinations thereof. In some aspects, the C4-dicarboxylic acid is malic acid, succinic acid, or fumaric acid, or combinations thereof. In some aspects, the C4-dicarboxylic acid is malic acid or fumaric acid, or a combination of malic acid and fumaric acid. In some aspects, the C4-dicarboxylic acid is malic acid.
In any of these aspects, the host cell produces (and/or is capable of producing) a C4-dicarboxylic acid at a yield of at least than 10%, e.g., at least than 20%, at least than 30%, at least than 40%, at least than 50%, at least than 60%, at least than 70%, at least than 80%, or at least than 90%, of theoretical.
In any of these aspects, the recombinant host has an C4-dicarboxylic acid volumetric productivity (e.g., malic acid volumetric productivity) greater than about 0.1 g/L per hour, e.g., greater than about 0.2 g/L per hour, 0.5 g/L per hour, 0.6 g/L per hour, 0.7 g/L per hour, 0.8 g/L per hour, 0.9 g/L per hour, 1.0 g/L per hour, 1.1 g/L per hour, 1.2 g/L per hour, 1.3 g/L per hour, 1.5 g/L per hour, 1.75 g/L per hour, 2.0 g/L per hour, 2.25 g/L per hour, 2.5 g/L per hour, or 3.0 g/L per hour; or between about 0.1 g/L per hour and about 2.0 g/L per hour, e.g., between about 0.3 g/L per hour and about 1.7 g/L per hour, about 0.5 g/L per hour and about 1.5 g/L per hour, about 0.7 g/L per hour and about 1.3 g/L per hour, about 0.8 g/L per hour and about 1.2 g/L per hour, or about 0.9 g/L per hour and about 1.1 g/L per hour.
The recombinant host cells may be cultivated in a nutrient medium suitable for production of the C4-dicarboxylic acid transporter, malate dehydrogenase, or pyruvate carboxylase using methods well known in the art, as described below.
The C4-dicarboxylic acid transporter, malate dehydrogenase, and pyruvate carboxylase, and activities thereof, can be detected using methods known in the art. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); and Hanai et al., Appl. Environ. Microbiol. 73:7814-7818 (2007)).

Methods

The present invention also relates to methods of using the recombinant host cells described herein for the production of a C4-dicarboxylic acid. In one aspect, the invention embraces a method of producing a C4-dicarboxylic acid (e.g., malic acid), comprising: (a) cultivating any one of the recombinant host cells described herein (e.g., any host cell with C4-dicarboxylic acid transporter activity, and optionally, malate dehydrogenase activity, and/or pyruvate carboxylase activity) in a medium under suitable conditions to produce the C4-dicarboxylic acid; and (b) recovering the C4-dicarboxylic acid. In one aspect, the invention embraces a method of producing a C4-dicarboxylic acid (e.g., malic acid), comprising: (a) cultivating in a medium any one of the recombinant host cells described herein, wherein the host cell comprises a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter; and optionally, a heterologous polynucleotide encoding a malate dehydrogenase, and/or a heterologous polynucleotide encoding a pyruvate decarboxylase under suitable conditions to produce the C4-dicarboxylic acid; and (b) recovering the C4-dicarboxylic acid. In one aspect, the medium is a fermentable medium.
In one aspect of the methods, the C4-dicarboxylic acid (e.g., malic acid) is produced at a titer greater than about 10 g/L, e.g., greater than about 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, 210 g/L, 225 g/L, 250 g/L, 275 g/L, 300 g/L, 325 g/L, 350 g/L, 400 g/L, or 500g/L; or between about 10 g/L and about 500 g/L, e.g., between about 50 g/L and about 350 g/L, about 100 g/L and about 300 g/L, about 150 g/L and about 250 g/L, about 175 g/L and about 225 g/L, or about 190 g/L and about 210 g/L.
In one aspect of the methods, the amount of produced C4-dicarboxylic acid (e.g., malic acid) is at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, or at least 100% greater compared to cultivating the host cell without the polynucleotide that encodes the C4-dicarboxylic acid transporter under the same conditions.
In some aspects of the methods, the C4-dicarboxylic acid is selected from malic acid, succinic acid, oxaloacetic acid, malonic acid, and fumaric acid. In one aspect, the C4-dicarboxylic acid is malic acid.
The recombinant C4-dicarboxylic acid can be optionally recovered from the fermentation medium using any procedure known in the art (see, for example, WO 1998/022611 and U.S. 7,601,865 ) including, but not limited to, chromatography (e.g., size exclusion chromatography, adsorption chromatography, ion exchange chromatography), electrophoretic procedures, differential solubility, osmosis, distillation, extraction (e.g., liquid-liquid extraction), pervaporation, extractive filtration, membrane filtration, membrane separation, reverse, or ultrafiltration. In one example, the C4-dicarboxylic acid is recovered from other material in the fermentation medium by filtration.
In some aspects of the methods, the recombinant C4-dicarboxylic acid before and/or after being optionally purified is substantially pure. With respect to the methods of producing a C4-dicarboxylic acid (or a specific C4-dicarboxylic acid thereof, such as malic acid), "substantially pure" intends a recovered preparation of the C4-dicarboxylic acid that contains no more than 15% impurity, wherein impurity intends compounds other than C4-dicarboxylic acids. In one variation, a preparation of substantially pure C4-dicarboxylic acid is provided wherein the preparation contains no more than 25% impurity, or no more than 20% impurity, or no more than 10% impurity, or no more than 5% impurity, or no more than 3% impurity, or no more than 1% impurity, or no more than 0.5% impurity.
Suitable assays to test for the production of C4-dicarboxylic acids for the methods of production and host cells described herein can be performed using methods known in the art. For example, the final C4-dicarboxylic acid product (e.g., malic acid), and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of C4-dicarboxylic acid in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual sugar in the fermentation medium (e.g., glucose) can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775 -779 (2005)), or using other suitable assay and detection methods well known in the art.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Strains

Aspergillus clavatus NRRL1 and Aspergillus fumigatus (Sartorya fumigata) Af293 were used as the source C4-dicarboxylic acid transporter genes. Aspergillus oryzae NRRL 3488 (or ATCC 56747) was used as a source of a pyruvate carboxylase gene, a malate dehydrogenase gene, and for production of the C4-dicarboxylic acids.

Media

YEG medium was composed of 20 g glucose, 5 g yeast extract, and deionized water to 1 liter.
COVE plates were composed of 1 M sucrose, 2% COVE salt solution, 10 mM acetamide, 15 mM CsCl, and 25 g/l Agar Noble.
COVE salt solution was composed of 26 g KCI, 26 g MgSO₄·7H₂O, 76 g KH₂PO₄, 50 ml of COVE trace elements solution, and deionized water to 1 liter.
COVE trace elements solution was composed of 0.04 g Na₂B₄O₇·10H₂O, 0.04 g CuSO₄·5H₂O, 1.2 g FeSO₄·7H₂O, 0.7 g MnSO₄·H₂O, 0.8 g Na₂MoO₂·2H₂O, 10 g ZnSO₄·7H₂O and deionized water to 1 liter.
Seed medium was composed of 40 g glucose, 6 g Bacto-peptone, 750 mg KH₂PO₄, 750 mg K₂HPO₄, 100 mg MgSO₄·7H₂O, 100 mg CaCl₂·H₂O, 5 mg FeSO₄·7H₂O, 5 mg NaCl, and deionized water to 1 liter.
Seed medium B is composed of 30 g glucose, 3 g Bacto Peptone, 560 mg KH₂PO_4, 560 mg K₂HPO₄, 925 mg NaH₂PO₄·H₂O, 820 mg Na₂HPO₄, 75 mg MgSO₄·7H₂O, 75 mg CaCl₂·H₂O, 0.75 ml of 1000X Micronutrient Solution, and deionized water to 1 liter.
Acid production medium C is composed of 100 g glucose, 80 g CaCO₃, 6 g Bacto Peptone, 150 mg KH₂PO₄, 150 mg K₂HPO₄, 100 mg MgSO₄·7H₂O, 100 mg CaCl₂·H₂O, 1 ml 1000X Micronutrient Solution, and deionized water to 1 liter.
1000X Micronutrient Solution is composed of 5 g NaCl, 5 g FeSO₄·7H₂O, 1 g citric acid, and deionized water to 1 liter.
PDA plates are composed of 39 g/l potato dextrose agar.

Example 1: Cloning of the Aspergillus clavatus NRRL1 C4-dicarboxylic acid transporter gene and construction of expression vector pShTh120AcC4T

The 1179 bp C4-dicarboxylic acid transporter gene acc4t (ACLA_058030) was synthetically constructed into pAcC4T (Figure 1; DNA2.0, Menlo Park, CA, USA). The acc4t gene was amplified from pAcC4T using primers 069735 and 069736 shown below.

Primer 069735:
5'-GTGTGATAGAACATCGTCCATAATGTTCGAAAATCG-3' (SEQ ID NO: 5)
Primer 069736:
5'-GTCAGTCACCTCTAGTTAATTAACTAGTCTGCAGCATCCTCATC-3' (SEQ ID NO: 6)

The PCR reaction mixture was composed of 50 ng pAcC4T template, 200 µM dNTP mixture, 50 pM primer 069735, 50 pM primer 069736, 1X Pol1 reaction buffer (New England Biolabs, MA, USA), and 1 unit Vent Polymerase (New England Biolabs) and deionized water to 50 µl. The PCR reaction was incubated in an EPPENDORF MASTERCYCLER® (Eppendorf Scientific Inc., Westbury, New York, USA) programmed for 1 cycle at 94°C for 3 minutes; 35 cycles at 94°C for 15 seconds, 59°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 5 minutes. The PCR product was purified by 1% agarose gel electrophoresis in TAE buffer (50 mM Tris base-50 mM acetate-0.1 mM disodium EDTA) and purified using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia, CA, USA).
Plasmid pShTh60 (Figure 2; see also PCT Application No. PCT/US10/47002 , entitled "Methods for Improving Malic Acid Production in Filamentous Fungi" filed August 27, 2010) was digested with Sex Al and Pac I then separated by 0.8% agarose gel electrophoresis in TBE buffer (10.8 g/L Tris Base, 5.5 g/L Boric acid, 2 mM EDTA, pH 8.0) and purified using a QIAQUICK® Gel Extraction Kit. The purified PCR product above was then inserted into the digested pShTh60 using an In-Fusion™ Cloning Kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions, resulting in pShTh120AcC4T (Figure 3). Plasmid pShTh120AcC4T was isolated using a QIAfilter Maxi Plasmid Isolation Kit (QIAGEN Inc., Valencia, CA, USA). DNA sequence analysis was used to confirm the integrity of the acc4t coding sequence using primers 996270 and 065067 shown below using an ABI3130XL DNA Analyzer (Applied Biosystems, Inc., Foster City, CA, USA) and the primer walking technique with dye-terminator chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60).

Primer 996270:
5'-CTATAGCGAAATGGATTGATTGTCT-3' (SEQ ID NO: 7)
Primer 065067:
5'-TGACCTTCCACGCTGACCAC-3' (SEQ ID NO: 8)

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the Aspergillus clavatus acc4t gene are shown in Figure 4. The genomic coding sequence of 1179 bp (including stop codon) encodes a polypeptide of 392 amino acids with a predicted mass of 43.4 kDa and an isoelectric pH of 7.85. The gene contains no introns. Using the Vector NIT® program (Invitrogen, CA, USA), a signal peptide of 52 residues was predicted, resulting in a predicted mature protein containing 340 amino acids.

Example 2: Cloning of the Aspergillus fumigates Af293 C4-dicarboxylic acid transporter gene and construction of expression vector pShTh121AfC4T

The 1182 bp C4-dicarboxylic acid transporter gene sequence afc4t (AFUA_8G04630) was synthetically constructed into pAfC4T (Figure 5; DNA2.0). The afc4t gene was amplified from pAfC4T using primers 069737 and 069738 shown below.

Primer 069737:
5-GTGTGATAGAACATCGTCCATAATGTTCAACGATCATGATCA-3'(SEQ ID NO: 9)
Primer 069738:
5'-GTCAGTCACCTCTAGTTAATTAATTAATCTAGCACATCCTCGTC-3' (SEQ ID NO: 10)

The PCR reaction mixture was composed of 50 ng pAtC4T template, 200 µM dNTP mixture, 50 pM primer 069737, 50 pM primer 069738, 1X Pol1 reaction buffer, 1 unit Vent Polymerase and deionized water to 50 µl. The PCR reaction was incubated in an EPPENDORF MASTERCYCLER® programmed for 1 cycle at 94°C for 3 minutes; 35 cycles at 94°C for 15 seconds, 59°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 5 minutes. The PCR product was purified by 1% agarose gel electrophoresis in TAE buffer (50 mM Tris base-50 mM acetate-0.1 mM disodium EDTA) and purified using a QIAQUICK® Gel Extraction Kit.
Plasmid pShTh60 (Figure 2) was digested and purified as described above. The purified PCR product above was then inserted into the digested pShTh60 using an InFusion Cloning Kit according to the manufacturer's instructions resulting in plasmid pShTh121AfC4T (Figure 6). Plasmid pShTh121AfC4T was isolated using a QIAfilter Maxi Plasmid Isolation Kit. DNA sequence analysis was used to confirm the integrity of the afc4t coding sequence using primers 996270 and 065067 as described above.
The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of the Aspergillus fumigates afc4t gene are shown in Figure 7. The genomic coding sequence of 1182 bp (including stop codon) encodes a polypeptide of 393 amino acids with a predicted mass of 43.8 kDa and an isoelectric pH of 7.30. The gene contains no introns.

Example 3: Transformation of expression vector fragments of pShTh120AcC4T and pShTh121AfC4T into Aspergillus oryzae NRRL3488 (ShTh1200 and ShTh1210)

Protoplast preparation and transformation of Aspergillus oryzae NRRL3488 were performed by inoculating approximately 2 X 10⁷ spores into 100 ml YEG medium and incubating the flask at 27°C for 16-18 hours at 140 rpm. Mycelia were collected by pouring the culture through a sterile funnel lined with MIRACLOTH® (Calbiochem, San Diego, CA, USA) and rinsing with 50 ml of 0.7 M KCI. The washed mycelia were resuspended in a 125 ml flask with 20 ml of protoplasting solution composed of 5 mg of GLUCANEX^™ (Novozymes A/S, Bagsværd, Denmark) and 0.5 mg of chitinase (Sigma Chemical Co., St. Louis, MO, USA) per ml of 0.7 M KCI (filter sterilized) and incubated at 34°C, for 30 minutes with mixing at 80 rpm. The protoplasting solution was poured through a sterile funnel lined with MIRACLOTH® and rinsed with 50 ml of STC buffer (1 M sorbitol-10 mM Tris-HCl pH 6.5-10 mM CaCl₂). The flow-through was collected in two 50 ml polypropylene tubes. The tubes were spun in the centrifuge at 1300 x g for 10 minutes at room temperature. The supernatant was discarded and the protoplast pellet was resuspended in 20 ml of STC buffer. The protoplasts were washed by two rounds of resuspending the pellet in 20 ml of STC and centrifugation at 1300 x g for 10 minutes at room temperature. The final pellet was resuspended in 2 ml of STC. The protoplasts were counted by removing a 10 µl sample and counting them in a haemocytometer (VWR, West Chester, PA, USA). The volume was adjusted with STC to obtain a protoplast concentration of 2x10⁷ per ml.
The plasmid vectors were prepared for transformation by restriction digestion with Pme I. The approximately 5 kb expression cassette from each construct was separated from the vector sequences by 0.8% agarose gel electrophoresis in TBE buffer and purified using a QIAQUICK® Gel Extraction Kit.
Four transformation reactions were prepared for each expression vector. For each reaction, a 100 µl solution of protoplast preparation was transferred to a 12 ml polypropylene tube, to which was added 2-5 µg of restriction digested plasmid vector above and 250 µl of polyethylene glycol solution (60% w/v polyethylene glycol (PEG), 10 mM Tris 6.5, 10 mM CaCl), followed by gentle mixing and incubation at 37°C for 30 minutes. Each transformation reaction was diluted with 6 ml STC, followed by three separate aliquots onto COVE plates. Each plate was then incubated at 34°C for 7-10 days. The resulting transformants were transferred to individual COVE plates and incubated at 34°C for 5 days. Spore stocks were prepared by collecting the spores in 0.1 % TWEEN® 80. Cultures were stored by preparing a glycerol stock of each (800 µl spore stock, 200 µl 0.1 % TWEEN® 80) and frozen at -80°C. Transformants containing the expression vector fragment of pShTh120AcC4T were designated ShTh1200. Transformants containing the expression vector fragment of pShTh121AfC4T were designated ShTh1210.

Example 4: Production of malic acid in shake flask cultures of Aspergillus oryzae transformants containing expression vector fragments of pShTh120AcC4T and pShTh121AfC4T (ShTh1200 and ShTh1210)

Spores from transformants ShTh1200 and ShTh1210 described above and Aspergillus oryzae NRRL 3488 as a control were plated onto individual COVE plates and allowed to sporulate at 34°C for 5 to 7 days. Spores were collected in 0.1% TWEEN® 80 and counted using a hemacytometer. Seed cultures were prepared in 250 ml flasks containing 100 ml of seed medium B and inoculated with 2 x 10⁸ total spores. Seed cultures were grown for approximately 17 hours at 30°C with shaking at 200 rpm. Acid production cultures were prepared in 250 ml unbaffled flasks containing 50 ml of acid production medium C and 3 ml of the 17 hour seed cultures. Cultures were incubated at 30°C with shaking at 200 rpm for 2-10 days.
Quantitation of malic acid for the shake flask culture transformants was performed by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) using an 1200 Series Binary LC System and 1200 Series Diode Array Detector (DAD) (Agilent Technologies, Santa Clara, CA USA). Reverse phase separation was performed using an Aqua 5µ C18 125Å 205 x 4.6 mm ID column and AQ C18 4 x 3.0 mm Security Guard Cartridge (Phenomenex, Inc., Torrance, CA, USA). The mobile phase consisted of 10% methanol (HPLC grade) and 90% 145 mM phosphate pH 1.5 buffer.
Whole culture samples were removed and diluted 1:10 in HPLC Running Buffer composed of 850 ml of 64 mM phosphate buffer and 150 ml of methanol pH 1.65. The samples were then filtered through a 25 mm 0.45 micron polyethersulfone membrane (Whatman, Florham Park, NJ, USA) and 1.5 ml of the filtrates were placed into a HPLC vial for acid analysis. The remaining amount of the shake flask cultures were filtered through 3 layers of cheese cloth and rinsed three times with 10 volumes of double distilled sterile water to remove insoluble CaCO₃. Cell pellets were harvested from the cheese cloth, placed into a 15 ml culture tube and stored at -20°C.
RP-HPLC was performed using an injection volume of 10 µl at a flow rate of 0.7 ml/minute (isocratic) with a column temperature of 25°C and run time of 11 minutes. Detection was set at 210 nm, 8 nm bandwidth, with the reference at 360 nm, 40 nm bandwidth. The void time was determined to be 3.8 minutes. The quantitative capabilities of the reverse phase method were determined for malic acid by performing replicate injections of serially diluted malic acid standards with concentrations ranging from 49.2-3.93 mM. The relative standard deviation for (RSD) for replicate injections was ≤5%. Malic acid shows R²≥ 0.9999.
Aspergillus oryzae transformants containing pShTh120AcC4T (strains ShTh1200) showed an improvement in malic acid production over the Aspergillus oryzae NRRL 3488 control strains and comparable malic acid production to Aspergillus oryzae ShTh1040 strains (see PCT Application No. PCT/US10/47002, filed August 27, 2010 ). Aspergillus oryzae transformants containing pShTh121AfC4T (strains ShTh1210) showed a slight improvement in malic acid production over the Aspergillus oryzae NRRL 3488 control strains and lower malic acid production compared to Aspergillus oryzae ShTh1040 strains.

Example 5: Fermentation of Aspergillus oryzae transformants containing expression vector fragments of pShTh120AcC4T (ShTh1200)

Aspergillus oryzae transformants above and control transformant Aspergillus oryzae ShTh1040 (see PCT Application No. PCT/US10/47002, filed August 27, 2010 ) were grown for approximately 7 days at 32°C on PDA plates. A 5-6 ml volume of sterile 50 mM sodium phosphate buffer (pH 6.8) containing 0.1% TWEEN® 80 was added to each plate and spores were suspended by scraping with an inoculating loop. Each suspension was transferred by pipette to a 50 ml conical tube. For each tube, 25 ml of sterile sodium phosphate buffer was added to a 500 ml unbaffled flask containing 75 ml of seed medium, which was then inoculated with 2 ml of spore suspension. The flasks were then incubated at 32°C and 180 rpm for about 24 hours. The seed flasks are combined to supply the 144 ml inoculum required per tank.
Three-liter fermentors containing 1.8 liters of medium were individually inoculated by introducing 144 ml (8%) of the seed culture broth from the combined seed flasks of either an Aspergillus oryzae pShTh120AcC4T transformant or an Aspergillus oryzae ShTh1040 transformant. The fermentors were equilibrated at 32 ± 0.1°C and stirred at 500 rpm. Inlet air flow was maintained at 1 v/v/m. Samples were withdrawn daily and analyzed for malic acid production, and the fermentations were completed after approximately 7 days.
Quantitation of malic acid in the fermentations was performed as described in Example 4. The relative malic acid titer of Aspergillus oryzae pShTh120AcC4T (ShTh1200) transformants were comparable to the Aspergillus oryzae ShTh1040 transformants, indicating that the Aspergillus oryzae pShTh120AcC4T transformants outperform the Aspergillus oryzae NRRL 3488 control (which lack the overexpressed C4-dicarboxylic acid transporter gene) based on ShTh1040 and NRRL 3488 comparisons previously described.

SEQUENCE LISTING

<110> Novozymes, Inc. Luttringer, Sheryl Yaver, Debbie
<120> Methods for Improved C4 Dicarboxylic Acid Production In Filamentous Fungi
<130> 11772-WO-PCT
<150> US 61/357,007
<151> 2010-06-21
<160> 16
<170> PatentIn version 3.5
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Claims

An Aspergillus oryzae host cell comprising a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter, wherein the transporter is selected from:
(a) a C4-dicarboxylic acid transporter having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2 or 4;

(b) a C4-dicarboxylic acid transporter encoded by a polynucleotide that hybridizes under high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, 3, or the full-length complementary strand thereof; and

(c) a C4-dicarboxylic acid transporter encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or 3,
wherein the Aspergillus host cell secretes increased levels of C4-dicarboxylic acid compared to the host cell without the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter when cultivated under the same conditions.
The host cell of claim 1, wherein the C4-dicarboxylic acid transporter comprises or consists of the mature polypeptide of SEQ ID NO: 2 or 4.
The host cell of any one of claim 1-2, wherein the mature polypeptide of SEQ ID NO: 2 is amino acids 53 to 392 of SEQ ID NO: 2, and the mature polypeptide of SEQ ID NO: 4 is amino acids 1 to 393 of SEQ ID NO: 4.
The host cell of any one of claims 1-3, wherein the C4-dicarboxylic acid transporter comprises or consists of SEQ ID NO: 2 or 4.
The host cell of any one of claims 1-4, wherein the heterologous polynucleotide encoding the C4-dicarboxylic acid transporter is operably linked to a promoter foreign to the polynucleotide.
The host cell of any one of claims 1-5, wherein the host cell further comprises a heterologous second polynucleotide encoding a malate dehydrogenase.
The host cell of claim 6, wherein the heterologous second polynucleotide encoding a malate dehydrogenase is operably linked to a promoter foreign to the polynucleotide.
The host cell of any one of claims 1-7, wherein the host cell further comprises a heterologous third polynucleotide encoding a pyruvate carboxylase.
The host cell of claim 8, wherein the heterologous third polynucleotide encoding a pyruvate carboxylase is operably linked to a promoter foreign to the polynucleotide.
The host cell of any one of claims 1-9, wherein the host cell is capable of secreting an increased level of the C4-dicarboxylic acid of at least 25% compared to the host cell without the polynucleotide encoding the C4-dicarboxylic acid transporter when cultivated under the same conditions.
The host cell of any one of claims 1-10, wherein the C4-dicarboxylic acid is malic acid.
A method of producing a C4-dicarboxylic acid, comprising:
(a) cultivating the host cell of any one of claims 1-11 in a medium; and

(b) recovering the C4-dicarboxylic acid.
A method for increasing C4-dicarboxylic acid production, comprising:
(a) transforming into a host cell a heterologous polynucleotide encoding a C4-dicarboxylic acid transporter, resulting in the host cell of any one of claims 1-11;

(b) cultivating the transformed host cell in a medium; and

(c) recovering the C4-dicarboxylic acid.